Hook2, a microtubule-binding protein, interacts with Par6α and controls centrosome orientation during polarized cell migration.

Polarity protein complexes function during polarized cell migration and a subset of these proteins localizes to the reoriented centrosome during this process. Despite these observations, the mechanisms behind the recruitment of these polarity complexes such as the aPKC/PAR6α complex to the centrosome are not well understood. Here we identify Hook2 as an interactor for the aPKC/PAR6α complex that functions to localize this complex at the centrosome. We first demonstrate that Hook2 is essential for the polarized Golgi re-orientation towards the migration front. Depletion of Hook2 results in a decrease of PAR6α at the centrosome during cell migration, while overexpression of Hook2 in cells induced the formation of aggresomes with the recruitment of PAR6α, aPKC and PAR3. In addition, we demonstrate that the interaction between the C-terminal domain of Hook2 and the aPKC-binding domain of PAR6α localizes PAR6α to the centrosome during cell migration. Our data suggests that Hook2, a microtubule binding protein, plays an important role in the regulation of PAR6α recruitment to the centrosome to bridge microtubules and the aPKC/PAR complex. This data reveals how some of the polarity protein complexes are recruited to the centrosome and might regulate pericentriolar and microtubule organization and potentially impact on polarized migration.

Evidence for a molecular link between the tuberous sclerosis complex and the Crumbs complex.

In human, mutations in tuberous sclerosis complex protein 1 or 2 (TSC1/2 or hamartin/tuberin) cause tuberous sclerosis characterized by the occurrence of multiple hamartomas. On the other hand, mutations in the Crumbs homolog-1 (CRB1) gene cause retinal degeneration diseases including Leber congenital amaurosis and retinitis pigmentosa type 12. Here we report, using a two-hybrid assay, a direct molecular interaction between TSC2 C-terminal part and PDZ 2 and 3 of PATJ, a scaffold member of the Crumbs 3 (CRB 3) complex in human intestinal epithelial cells, Caco2. TSC2 interacts not only with PATJ, but also with the whole CRB 3 complex by GST-pull down assays. In addition, TSC2 co-immunoprecipitates and co-localizes partially with PATJ at the level of the tight junctions. Furthermore, depletion of PATJ from Caco2 cells induces an increase in mammalian Target Of Rapamycin Complex 1 (mTORC1) activity, which is totally inhibited by rapamycin. In contrast, in the same cells, inhibition of phosphoinositol-3 kinase (PI-3K) by wortmannin does not abolish rpS6 phosphorylation. These functional data indicate that the Crumbs complex is a potential regulator of the mTORC1 pathway, cell metabolism and survival through a direct interaction with TSC1/2.

CRB3 binds directly to Par6 and regulates the morphogenesis of the tight junctions in mammalian epithelial cells.

Crumbs is an apical transmembrane protein crucial for epithelial morphogenesis in Drosophila melanogaster embryos. A protein with all the characteristics for a Crumbs homologue has been identified from patients suffering from retinitis pigmentosa group 12, but this protein (CRB1) is only expressed in retina and some parts of the brain, both in human and mouse. Here, we describe CRB3, another Crumbs homologue that is preferentially expressed in epithelial tissues and skeletal muscles in human. CRB3 shares the conserved cytoplasmic domain with other Crumbs but exhibits a very short extracellular domain without the EGF- and laminin A-like G repeats present in the other Crumbs. CRB3 is localized to the apical and subapical area of epithelial cells from the mouse and human intestine, suggesting that it could play a role in epithelial morphogenesis. Indeed, expression of CRB3 or of a chimera containing the extracellular domain of the neurotrophin receptor p75NTR and the transmembrane and cytoplasmic domains of CRB3 led to a slower development of functional tight junctions in Madin-Darby canine kidney cells. This phenotype relied on the presence of CRB3 four last amino acids (ERLI) that are involved in a direct interaction with Par6, a regulator of epithelial polarity and tight junction formation. Thus, CRB3, through its cytoplasmic domain and its interactors, plays a role in apical membrane morphogenesis and tight junction regulation.

The scaffolding domain of caveolin 2 is responsible for its Golgi localization in Caco-2 cells.

In this work, we showed that in Caco-2 cells, a polarized cell line derived from human colon cancer that does not express caveolin 1 (Cav-1), there was no detectable expression of caveolin 2 (Cav-2). When Cav-2 was reintroduced in these cells, it accumulated in the Golgi complex. A chimera, in which the scaffolding domain of Cav-1 was replaced by the one from Cav-2, induced a prominent Golgi staining of Cav-1, strongly indicating that this domain was responsible for the accumulation of Cav-2 in the Golgi complex. Cav-2 was able to interact with Cav-1 in the Golgi complex but this interaction was not sufficient to export it from this compartment. Several chimeras between Cav-1 and 2 were used to show that surface expression of caveolin was necessary but not sufficient to promote caveolae formation. Interestingly, levels of incorporation of the chimeras into Triton insoluble rafts correlated with their ability to trigger caveolae formation raising the possibility that a critical concentration of caveolins to discrete domains of the plasma membrane might be necessary for caveolae formation.

hINADl/PATJ, a homolog of discs lost, interacts with crumbs and localizes to tight junctions in human epithelial cells.

dCrumbs is an apical organizer crucial for the maintenance of epithelial polarity in Drosophila (1). It is known that dCrumbs interacts with Discs lost (Dlt), a protein with four PDZ (PSD95/Discs Large/ZO-1) domains (2), and Stardust (Sdt), a protein of the MAGUK (membrane-associated guanylate kinase) family (3, 4). We have searched for potential homologs of Dlt in human epithelial cells and characterized one of them in intestinal epithelial cells. Human INAD-like (hINADl) contains 8 PDZ domains, is concentrated in tight junctions, and is also found at the apical plasma membrane. Overexpression of hINADl disrupted the tight junctions localization of ZO-1 and 3. We also identified a partial cDNA coding the transmembrane and cytoplasmic domains of a new human crumbs (CRB3) expressed in Caco-2 cells. This CRB3 was able to interact through its C-terminal end with the N-terminal domain of hINADl. Taken together, the data indicate that hINADl is likely to represent a Dlt homolog in mammalian epithelial cells and might be involved in regulating the integrity of tight junctions. We thus propose to rename hINADl PATJ for protein associated to tight junctions.

Role of the membrane-proximal O-glycosylation site in sorting of the human receptor for neurotrophins to the apical membrane of MDCK cells.

We have analyzed the respective roles of the stalk and/or the O-glycosylation sites in apical sorting by producing partially deleted mutants in this region of the human receptor for neurotrophins (P75(NTR)). The mere presence of O-glycosylations was not sufficient for efficient delivery to the apical surface since changing the stalk domain of P75(NTR) for the heavily O-glycosylated stalk from human decay-accelerating factor led to random distribution of the chimera. The presence of O-glycosylations, however, was a prerequisite for exit from the ER and protection from intracellular cleavage since a P75(NTR) containing the non O-glycosylated stalk of the human placental alkaline phosphatase was not transported to the cell surface but was cleaved and secreted from the basolateral side. Deletion of the membrane-proximal part of the stalk showed a more dramatic reversal of polarity of P75(NTR) than the deletion of the distal part. Furthermore, moving the first putative O-glycosylation site (T216) two amino acids away from the membrane resulted in a loss of apical polarity of P75(NTR), suggesting that an important clue for apical sorting resides in this part of the stalk. This loss of apical polarity paralleled a loss of association of P75(NTR) mutants with Lubrol rafts. These data indicate that the position of O-glycans in the proximal part of the stalk domain of P75(NTR) is crucial for apical sorting and may regulate association with apical rafts.